Chemistry

Proteolysis of insulin in the endoplasmic reticulum followed by cleavage of the C chain of pro-insulin to form insulin is a good example of posttranslational modification. This paper looks at the insulin molecule, with a particular reference to its primary, secondary, tertiary and quaternary structures. Features of the protein which enable the molecule to perform its biochemical functions are also considered as are the posttranslational modifications and cellular activity. Structurally, insulin is organized at 4 levels the primary, secondary, tertiary and quaternary levels. It was found that insulin has 2 chains, the  and the  chains which are held together by disulfide bonds. The secondary structure is largely held together by van der waals forces and salt bridges. The quaternary structure of insulin consists of hexamers that arise from the association between hydrophobic surfaces. The molecule is first synthesised as preproinsulin and cleaved to form proinsilin and finally insulin. Insulin has tyrosine kinase activity and this enables the molecule to attach very tightly to its receptors and induce a series of phosphorylations which finally lead to the translocation of GLUT receptors and uptake of glucose by the target cells. It also leads to activation of the MAPK system.

Insulin is a protein hormone that is made up of 2 polypeptide chains and which has a molecular weight of 6kDa. Produced by the  cells of the islets of Langerhans in the pancreas, insulin is an active promoter of anabolic processes in the body. In this regard, insulin is associated with enhancement of the rate of glycogenesis, protein, and fatty acid synthesis. Insulin also enhances the uptake of glucose into muscle and adipose tissues and thus has a potent hypoglycaemic effect. Besides the anabolic effect, insulin also exerts a catabolic effect and it does this by reducing the level of enzymes that are necessary for such cellular processes as gluconeogenesis (Ganong, 1999)

. This paper discusses the chemistry and biochemistry of insulin with a particular emphasis on the synthesis methods, modifications (amino acid replacements, deletions, additions) and their effects. The role of metal ions in activation of insulin as well as the biochemical mechanisms underlying the function of this hormone is also discussed in an in-depth manner (Berg, Tymoczko,  Stryer, 2008)

The Primary Structure of Insulin
. The primary structure of insulin consists of 2 polypeptide chains called the A chain and the B chain. The A chain consists of 21 amino acid residues while the B chain consists of 30 amino acid residues (table 1 and 2). The 2 polypeptide chains are joined to each other via 2 disulfide bonds. These bonds are formed between the cysteine residues located on the 7th position of both chains and between the cysteine residue located on the 20th position of the A chain (Cys 20) and the cysteine residue on the 19th position of the B chain (Cys 19) (Berg, Tymoczko,  Stryer, 2008)

However, there is a variation of the composition of the chains depending on the species.

Secondary Structure of Insulin
The secondary structure of insulin is formed as a result of hydrogen bonds between the N-H and CO groups which jut out from the peptide bonds of amino acids situated 3 or 4 residues further along. The amino acid residue coil to create short sections of alpha helix and this helps to make the insulin molecule more stable.
The A chain has 2 turns of alpha helix between the isoleucine residue at position 2 and the threonine residue at position 8 (A2 Ile  A8 Thr) and between the leucine residue at position 13 and the tyrosine residue at position 19 (A13 leu-A19 Tyr). These 2 sections are separated by a flat ribbon that allows the 2 alpha helices to remain adjacent to each other and permits the creation of Van der Waals forces between the side chains of A2 Ile and A19 Tyr (Berg, Tymoczko,  Stryer, 2008). 

On the other hand, the B chain has a V shape arising from the alpha helix formed between the serine residue at position 9 and the Cysteine residue at position 19 (B9 Ser-B19 Cys) and between glycine residues at positions 20 and 23 (B20 Gly  B23 Gly). This causes the C terminal residues B24 Phe and B26 Tyr to associate with B15 Leu and B11 Leu via Van der Waals forces.

Tertiary structure of Insulin
    The tertiary structure of insulin consists of disulfide bridges which occur between the thiol groups (SH) of Cysteine. From the primary structure, it can be seen that there are a total of 6 cysteine residues at positions 6, 7, 11 and 20 on the A chain and at positions 7 and 19 on the B chain. From figure 1, it is evident that 3 disulfide bonds are formed and one of these occur in the A chain between A6 Cys and A11 Cys. The other 2 are created between the A and B chains between A7 Cys and B7 Cys and between A20 Cys and A19 Cys. Besides these disulfide bonds, the tertiary structure of insulin is also composed of numerous Van der Waals forces and salt bridges.

Quaternary structure of Insulin
The quaternary structure of insulin consists of hexamers that arise from the association between hydrophobic surfaces. Six insulin molecules band together around 2 zinc ions to create the hexamers. This structure has a toroidal shaped (i.e. it is shaped like a doughnut) and results in granules. It is in this form that insulin is stored in the pancreatic B cells and secreted into the circulation. Insulin can also exist in dimmers (2 units) or monomers (single units). Reportedly, insulin exists as a monomer in the active state.

Synthesis of Insulin
Human insulin is initially synthesised as a 110 amino acid single chain called the preproinsulin. This molecule is then converted to proinsulin by removal of the signal peptide.

Preproinsulin has 19 additional residues which are not present in the proinsulin molecule. These residues form a hydrophobic region which plays a vital role in the cleavage of the molecule into proinsulin. According to Berg, Tymoczko,  Stryer, (2008), this hydrophobic region acts as a signal sequence and causes the preproinsulin to move into the endoplasmic reticulum (ER). Once here, the hydrophobic region is cleaved off to form the proinsulin molecule.

The proinsulin molecule differs from the insulin molecule in that it has a third chain called the C-chain.  As can be seen in the sequence above, the C chain is composed of 30 additional residues which are absent in the insulin molecule. Figure 5 below illustrates the location of this C chain. It joins the amino end of the A chain and the carboxyl end of the B chain. There is a high degree of sequence homology between the C chains of proinsulins of different species. A common feature is that all the C chains have at their carboxyl ends arginine and lysine molecules at their amino ends 2 consecutive arginine molecules. Lysine and arginine are both basic amino acids with a net positive charge. It is these positively charged ends which form the cleavage sites for the proteolytic enzymes that cleave off the C chain. The lysine and arginine molecules occur at positions 62 and 63 respectively on the A chain of the proinsulin molecule in porcines while the 2 arginie residues are found at the 32 and 31 at the amino end of the A chain.
Conversion to insulin involves removal of this chain and the formation of disulfide linkages between the A and B chains.

Briefly, cleavage of the C chain involves transportation of the proinsulin molecule to the Golgi apparatus and to secretory granules where proteolytic enzymes cleave off the chain at the described positions.  Cleavage of the chain is mediated by the prohormone convertase 1 and 2 and results into an insulin molecule that has the 2 chains. Cleavage of the chain is followed by the removal of 2 amino acids are by the enzyme carboxypeptidase E.

Activity of Insulin
The tyrosine kinase activity of insulin has been well documented. To fulfil its functions in the body, insulin first of all binds to particular receptors which are localized on the plasma membranes of the target cells. The attachment of insulin to these receptors is very tight and this is central to the activity of the hormone. The receptor for insulin is made up of 2  and 2  chains. The  chains are 135 kd whereas the  chains measure 95 kd. These receptors are integral membrane glycoproteins and they innervate the plasma membrane, with obvious intracellular and extra cellular domains. In general,  chains occur principally on the extra cellular portion of the plasma membrane whilst the  chains are to be found inside the plasma membrane (Ottensmeyer FP et al, 2000).

Both chains originate from one main chain precursor which has a total of 1382 residues. The precursor molecule is made up of signal sequence, and the  and  chain sequences in that order. The  and  sequences are however separated by a tetra peptide which is composed of basic amino acids notably lysine and arginine. The common sequence of this tetra peptide is arginine-lysine-arginine and a final arginine molecule. This sequence plays an important role in the signal transduction pathway since it is recognized by the processing enzymes. The signal sequence is cleaved off and this activates the entire pathway. When activated, the insulin receptor acts as an enzyme and it phosphorylates tyrosine residues in the target proteins (Ottensmeyer FP et al, 2000).
On the cytoplasmic region of the receptor there are also tyrosine domains which occur largely in the B chains while the insulin biding sites are on the  chain on the portion of the membrane which is found on the outside of the cell. When insulin attaches to the receptor on the extra cellular the catalytic activity of the tyrosine kinase enzyme is turned on, leading to the phosphorylation of 2 tyrosine residues (Ottensmeyer FP et al, 2000).  
This is followed by the phosphorylation of the insulin receptor substrates (RS-IRS6). These are docking proteins and once they become phosphorylated, they attach to other kinases and activate them. Phosphatidyl inositol 3 kinase is the enzyme which is most often stimulated by the actions of the IRS proteins and these lead on to further phosphorylation which activate adaptor proteins. The most common adaptor protein which is turned on by the activity of the phosphatidyl inositol 3 kinase is the growth factor receptor binding protein 2 and this transduces this signal onto a guanine releasing factor. The latter molecule subsequently leads to the activation of the ras which is a GTP binding protein and mitogen-activated protein kinase (MAPK) system. These series of reactions constitute the second messenger system of insulin. The phosphorylation cause many effects, the most important of which is the movement of glucose transporters such as GLUT to the cell membrane and this causes the cells to increase their uptake of glucose (Nolte  Karam, 2007 Ottensmeyer FP et al, 2000).
Insulin is usually released from the B cells upon stimulation by glucose and mannose, amino acids such as arginine and leucine, and hormones such as the glucagons-like-polypeptide 1 (GLP-1). Release of insulin into the bloodstream can also be induced by certain drugs such as sulfonylurea (Dunning et al, 2005).

Figure  SEQ Figure  ARABIC 5 Control of insulin secretion
 Source Bertram (2008)

The transporters of Glucose


Source Katzung, (2007)
Insulin is processed in the endoplasmic reticulum where cleavage of the C chain from the proinsulin molecule occurs to form insulin. This is a prime example of posttranslational modification in eukaryotes. This paper looked at the insulin molecule, with a particular reference to its primary, secondary, tertiary and quaternary structures. Features of the protein which enable the molecule to perform its biochemical functions were also considered as were the posttranslational modifications and cellular activity. Structurally, insulin is organized at 4 levels the primary, secondary, tertiary and quaternary levels.

Obesity can be defined as a condition whereby an individual has an abnormally high proportion of body fat. It is considered a disease by the World Health Organization (WHO). It can also be defined clinically where an obese person is said to be having a BMI Body Mass Index- weight (kg) height(m)2 of greater than 30 while overweight is a BMI of 25-29.9. It is possible to have an individual who is overweight but not obese. This is so because someone overweight can be said to be having excess weight which might be a result of muscle, bone, fat andor body water. About two-thirds of adults in the United States are overweight, and almost one-third are obese, according to data from the National Health and Nutrition Examination Survey (NHANES) 2001 to 2004. The rising obesity rates have resulted in numerous health complications chiefly nutrition related diseases over the last few decades.
The above few statistics coupled with the fact that obesity is becoming more and more widespread has generated interest in obesity. Obesity has come to be regarded as a social problem. Obesity prevalence has lead to an urgent need for evidence to support advocacy and for corrective policies and intervention programs targeting an array of societal sectors (Kumanyika 2007).
A simplistic cause of obesity can be said to be consuming more calories than one is expending thus accumulating fat as a result. In a 2006 Pew Research poll asking Americans why the nation consumed so much junk food, convenience was the most popular answer. The same poll revealed that more children can identify Ronald McDonald than Santa Claus (Pew research poll qtd. in waterson 2009)Causes of obesity are more complex than this. It has become synonymous with economic and technological advancements. Obesity can be said to have a wide range of causes. Genes and individual psychobiology is one cause that has been greatly investigated over the years. Obesity can also be said to be caused by processes indispensable to human survival and interaction.
Obesity has become an important field of study today because the increase in nutrition related chronic disease morbidity and mortality have brought about significant burdens to families, communities and health care systems. Diseases associated with overweight and obesity include diabetes, coronary heart disease, high blood cholesterol, stroke, hypertension, gall bladder disease, osteoarthritis, sleep apnea and some forms of cancer. Implications brought about by being obese have immense consequences on the global as well as the American economy. According to the weight control information network, the estimated total cost of implications arising from being obese and overweight is  117 billion. Quite an astronomical figure by any standards (Weight control information Network 2007).
Knowing obesity causes will not necessarily lead to solutions. This is so because it is practically impossible to remove high calorie foods from our diets, do away with automobiles or return to our lean hunter gatherer eras. All these factors are deeply embedded in our lifestyles. The way to go would be to shift some environmental factors in a direction more favorable to achieving energy balance while also fostering individual behavior changes in the same direction (Kumanyika 2007).
Projections by Wang and Beydoun show that 75 percent of adults will be overweight (body mass index _25 kgm2) or obese (body mass index _30 kgm2) and that 41 percent will be obese by 2015 (Wang and Beydoun 2007).
These are truly shocking statistics that have contributed immensely to raising awareness and making studies on obesity and its probable solutions an immediate and important priority.
It was thought for a long time that weight issues and obesity was predisposed to people of high social standing. On the contrary, recent studies indicate that the low cost of high-calorie foods that are easy to over consume predisposes to obesity among the poor. In fact in high income countries, the gap in obesity levels between the high and low socioeconomic strata may be decreasing (Kumanyika 2007). Many people tend to purchase foods that offer the greatest caloric content for the price when hunger lurks and money is tight. This according to Plotkin is the very real link between being poor and being overweight and obese (Plotkin 2009).
Another interesting factor that has been linked to obesity is the built environment. Built environment refers to factors such as community design, location of retail food outlets, recreational facilities and the transportation infrastructure, which determine the availability and convenience of options for physical activity and food acquisition. According to papa et al.changes in the built environment are potential strategies for corrective interventions to improve the eating and physical activity patterns of populations (Papa et al. qtd in Kumanyika 2007).
Chemistry factors in quite strongly or becomes relevant in obesity issues when the processed foods factor is considered. Processed foods contain elements that are known to cause cardiac problems, obesity, overweight problems and a host of other lifestyle diseases. An example of such elements is trans fats or trans-fatty acids. According to the natural health information centre, these are unnatural compounds which are known to be detrimental to health. They are created by industrial processes where hydrogen is added to liquid vegetable oil to make it more solid. (Americanheart.org 2009)
They are referred to as unsaturated because they have double bonds between adjacent carbon atoms. Saturated fats have no double bonds and all the spaces available are taken up by hydrogen atoms.
When a natural unsaturated fatty acid is hydrogenated, it is not possible to control where the hydrogen atoms are added to the structure. If both hydrogen atoms are added to the same side of the structure, it is referred to as Cis fats. These are naturally occurring and able to allow other chemicals and enzymes to bind to them. When one hydrogen atom adds to one side of the structure and the other atom to the other side, then a trans fat comes into being.
Trans fats do not exist naturally except with a few exceptions. Their structure is not crowded and so they do not accommodate other enzymes and molecules. Trans fats being straight can solidify at room temperature (National-health-information-centre.com 2008).
Trans fats raise the bad cholesterol levels (LDL) and lower the good cholesterol level (HDL). This increases the risk of developing heart diseases, stroke and type 2 diabetes not to mention the increased fatty accumulation and therefore obesity. They are present in fried foods, baked goods, stick margarines and shortenings (Americanheart.org).
Other studies are being conducted into relatively new compounds like 2-furoylmethyl aspartic acid, 2-furoylmethyl pyrrolidone carboxylic acid and 2-furoylmethyl lysine (furosine) which have been detected in stored dehydrated orange juice and tomato products to determine their exact identities and their contribution to the obesity endemic.
Current frontiers on fighting obesity remain largely new uncoordinated. Discrepancies in social facilities that promote healthy living habits should be addressed. Sensitization starting in schools should be carried out on the entire populace to promote healthy eating habits and promote increased physical activities. Integration of schools as a key partner in the fight against obesity would be a good step in the right direction as success in tackling obesity and overweight in childhood and adolescent would translate into success in dealing with obesity in later years. Though obesity prevention is still in its infancy, it is an emerging field with a clear mandate.

Obesity has become the second leading preventable cause of disease and death in the United States, second only to tobacco use (Wang and Beydoun 2007). This trend is on an upward trend unless serious and urgent mitigation measures are taken.
Obesity is caused by many factors mostly lifestyle related. In most people, high calorie intake without adequate physical activity has brought about adding of weight. In order to maintain a healthy weight, a balance must be observed between energy consumption through dietary intake and energy expenditure through metabolic and physical activity. Studies have also revealed that chemistry is an emerging factor that has a role to play in obesity. Trans fats which are chemical compounds produced in industry are the main culprits. Studies are ongoing to determine effects of new compounds detected in dehydrated orange juice and stored tomato products.